Double-stranded and single-stranded RNA molecules with 5′ triphosphates and their use for inducing interferon

ABSTRACT

Double-stranded and single-stranded RNA molecules, and their use in methods for inducing interferon are provided. The interferon induction provides anti-viral and other medically useful effects, such as anti-cancer effects. Also provided are methods for reducing or inhibiting interferon induction exhibited by such molecules, particularly siRNA and shRNA molecules produced in vitro.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a division of U.S. patent application Ser.No. 11/347,833 filed on 6 Feb. 2006 which in turn is related to andclaims priority under 35 U.S.C. §119(e) to U.S. provisional patentapplication Ser. No. 60/649,537 filed on 4 Feb. 2005. Each applicationis incorporated herein by reference.

The invention described herein was made with Government support undergrant number HL074704 from NHLBI of the National Institutes of Health.Accordingly, the United States Government may have certain rights inthis invention.

FIELD OF THE INVENTION

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated byreference, and for convenience are referenced in the following text byauthor and date and are listed alphabetically by author in the appendedbibliography.

The present invention relates to RNA molecules, includingdouble-stranded and single-stranded RNA molecules, and their use forinducing interferon. The present invention also relates to methods forcontrolling interferon induction by such molecules.

BACKGROUND OF THE INVENTION

Small interfering RNAs (siRNA) are potent reagents for directedpost-transcriptional gene silencing (Hannon, G. J., 2002). siRNAs aredouble-stranded molecules typically 21 to 25 nucleotides (nt) in length,which trigger RNA interference (RNAi), resulting in post-transcriptionalmessage degradation (Elbashir, S. M., et al., 2001) and inhibition ofviral propagation (Andino, R., 2003). RNAi has emerged as an immenselyimportant and popular method to elicit post-transcriptional,sequence-specific silencing of gene expression and is a major newgenetic tool for investigating mammalian cells. RNAi is initiated byexposing cells to dsRNA either via transfection or endogenousexpression. Long double-stranded (ds) RNAs are processed into siRNAs bydicer, a ribonuclease of the Rnase III family. These siRNAs form acomplex known as the RNA Induced Silencing Complex or RISC, whichfunctions in homologous target RNA destruction (Montgomery, M. K.,2004).

The use of exogenously supplied siRNAs for targeted RNA knockdowns hasbecome widespread (Elbashir, S. M., et al., 2001). The exogenous RNAscan be manufactured synthetically. However, when synthetic siRNAs areused for gene silencing, the costs can be substantial because ofvariations in siRNA efficacies. An alternative to chemically synthesizedsiRNAs are siRNAs produced by bacteriophage T7 RNA polymerase. ThesesiRNAs are made by in vitro transcription mediated by bacteriophagepromoters from linearized DNA templates. In vitro transcription usingbacteriophage T7 RNA polymerase has been shown to produce highly activesiRNAs (Sohail, M., et al., 2003; Donze, O. and Picard, D., 2002).

The interferon (IFN) system is one of the body's first lines of defenseagainst viruses (Samuel, C. E., 2004). IFN was discovered as anantiviral agent by Isaacs and Lindenmann during studies on virusinterference, where they observed that cells infected with influenzavirus secrete a factor that mediates the transfer of a virus-resistantstate active against both the inducing virus and other viruses as well(Samuel, C. E., 2004). Double-stranded RNA (dsRNA) is known to play animportant role in the IFN system (Samuel, C. E., 2001). It is known thatsynthetic dsRNAs and RNAs with double-stranded character produced duringviral infections have the capacity to be potent inducers of IFN(Stewart, W. E., 1979; Marcus, P. I., 1983).

The early recognition of invasive pathogens by innate sensing is themost important defense mechanism of the immune system (Beutler, B.,2004a; Boehme, K. W. and Compton, T., 2004). Viral infection ofmammalian cells results in activation of an innate immune response whichis mediated by interferons and cytokines that concomitantly inhibitviral replication (Malmgaard, L., 2004). Several Toll-Like Receptors(TLRs) have been identified in humans and mice and are known to beexpressed predominantly on cell types which are first to encounterintracellular pathogens (Boehme, K. W. and Compton, T., 2004). Doublestranded RNA (dsRNA), including the synthetic analog poly inosine-polycytosine (Poly IC), is known to activate TLR3, a cellular receptor thatrecognizes and initiates a potent anti-viral response by producinginterferons (Alexopoulou, L., et al., 2001). Similarly, single strandedRNA (ssRNA), which includes the genomes of several viral RNA species,has been shown to interact with and activate TLR7 and TLR8 (Lund, J. M.,et al., 2004; Diebold, S. S., et al., 2004; Heil, F., et al., 2004;Hornung, V., et al., 2005). dsRNAs can be easily distinguishedintracellularly as viral replication intermediates, however, it remainselusive how a simple ssRNA motif recognized by TLR7 and 8 is discernedby the cell to be either viral (exogenous) or endogenous in origin(Boehme, K. W. and Compton, T., 2004). Considering that TLRs are celltype specific and are present within unique localized intracellularcompartments, recognition of dsRNA and/or ssRNA offers an importantinnate defense mechanism against viral infection along with therecognition of CpG DNA motifs and/or envelope glycoproteins (Boehme, K.W. and Compton, T., 2004; Beutler, B., 2004b)

RNAi-mediated gene silencing in mammalian cells requires siRNAs ofsufficiently small size to circumvent potential sequence-independent,nonspecific changes in gene expression attributable to the induction oraction of interferons. Sledz, C. A., et al. (2003) found thattransfection of siRNAs results in interferon (IFN)-mediated activationof the Jak-Stat pathway and global upregulation of IFN-stimulated genes.The authors showed that by using cell lines deficient in specificcomponents mediating IFN action that the RNAi mechanism itself isindependent of the interferon system. The authors characterized theirfinding as showing the “broad and complicating effects” of siRNAs beyondthe selective silencing of target genes when introduced into cells.Similarly, Bridge, A. J., et al. (2003) reported that although siRNAswere thought to be too short to induce interferon expression, a commonlyused shRNA construct was found to induce an interferon response. Theauthors advise as a “simple precaution to limit the risk of inducing aninterferon response” to use the lowest effective dose of shRNA vector.

Although the anti-viral activities of interferons are well studied(Samuel, C. E., 2001), nobody has recognized in connection with RNAi theuses and advantages, as opposed to the risks, of interferon induction byRNAi molecules, independent of the RNAi effect, to provide anti-viraland other effects, such as anti-cancer effects. Moreover, until now,nobody is believed to have discovered the role of the triphosphate, inparticular the 5-triphosphate produced on RNAi molecules in vitro, forinducing interferon and eliciting anti-viral and other medically usefulresponses.

SUMMARY OF THE INVENTION

The present invention is believed to be first to show that the presenceof an initiating triphosphate on in vitro transcribed-RNAs can potentlyinduce interferon α and β, as well as elicit a strong,non-sequence-specific antiviral response to viral challenge.

The present invention relates in one aspect to double-stranded RNAmolecules, including RNAi molecules, and in another aspect tosingle-stranded RNA molecules, on which one or more triphosphates,preferably one or more 5′-triphosphates, are maintained in order toexploit the interferon induction properties of such molecules, in orderto provide anti-viral and other medically useful (e.g., anti-cancer)effects.

The present invention relates in one embodiment to a method for inducinginterferon in a cell, comprising exposing or introducing into the cellan effective amount of an RNAi molecule having one or moretriphosphates, preferably a 5′-triphosphate, wherein said RNAi moleculeinduces said interferon. The RNAi molecule also can have an anti-viraleffect, and preferably, is introduced into the cell prior to viralinfection, wherein the RNAi molecule inhibits or prevents viralinfection. The RNAi molecule also can have other medically usefuleffects, such as an anti-cancer effect.

In another embodiment, the invention provides a composition for inducinginterferon in a cell comprising an effective amount of an RNAi moleculehaving one or more triphosphates, preferably a 5′-triphosphate, whereinthe RNAi molecule can induce interferon in the cell. In a preferredembodiment, the RNAi molecule can also have an anti-viral or anti-cancereffect.

In another embodiment, the invention provides an anti-viral reagentcomprising an effective amount of an RNAi molecule having one or moretriphosphates, preferably a 5′-triphosphate, wherein the RNAi moleculein addition to inducing interferon also has an anti-viral effect. In oneembodiment, the anti-viral effect is the result of interferon induced bythe RNAi molecule in a non-sequence dependent manner. In anotherembodiment, the anti-viral effect is the result of a synergy between anRNAi effect mediated by the RNAi molecule (i.e., as a result of homologybetween the RNAi molecule and its target molecule) and an immuneresponse mediated by interferon induction.

In another embodiment, the invention provides a method for inducing ananti-viral response in a cell, comprising introducing into a cell aneffective amount of an RNAi molecule having one or more triphosphates,preferably a 5′-triphosphate, and which exhibits one of the aboveanti-viral effects.

The cell can be any cell and is preferably a eukaryotic or vertebratecell, more preferably a mammalian cell, and most preferably a humancell.

In a preferred embodiment, the RNAi molecule is an siRNA or an shRNA.

In another aspect, the present invention provides a method for inducinginterferon in a cell, comprising introducing into the cell an effectiveamount of a short single-stranded RNA (ssRNA) having one or moretriphosphates, preferably a 5′-triphosphate, wherein the ssRNA moleculeinduces interferon, and preferably also has an anti-viral or othermedically useful (e.g., anti-cancer) effect, as described above.

In a more preferred embodiment of each of the above embodiments, theRNAi molecule and ssRNA molecule are produced in vitro by a phagepolymerase. In a preferred embodiment, the phage polymerase is T7 RNApolymerase, T3 RNA polymerase or Sp6 RNA polymerase. In an even morepreferred embodiment, the polymerase is T7 RNA polymerase.

In the present invention, the 5′-triphosphate of an RNAi or ssRNAmolecule produced in vitro has been discovered to be an active inducerof interferon, as well as a potent anti-viral agent. On the other hand,the present invention also recognizes advantages of removing the 5′triphosphate from in vitro transcribed RNAi molecules, and thus reducingor inhibiting interferon induction. This additional aspect of theinvention should be useful for controlling, reducing or inhibitinginterferon induced during gene silencing using such RNAi molecules.

In yet another aspect, the present invention thus provides an in vitromethod for producing or synthesizing an RNAi molecule which reduces oralleviates the interferon response exhibited by a double-stranded,preferably an RNAi, molecule or ssRNA molecule produced in vitro, whilemaintaining the efficacy of the molecule. In one embodiment, the methodcomprises removing one or more 5′-triphosphates from the molecule,wherein the removal reduces or alleviates the interferon response whilemaintaining the efficacy of the molecule.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A are photographs showing anti-HSV-1 activities of T7 transcribedsiRNAs. The siRNA transfected HEK-293 cells were infected withHSV-1-EGFP. Top panel, mock or chemically synthesized siRNA transfectedsamples (anti-La#2 and anti-Ro #1); middle panel, T7 siRNA transfectedsamples; lower panel, the anti-La #2 siRNAs were prepared by T7 RNApolymerase using the Silencer siRNA Construction Kit (Ambion).

FIG. 1B are photographs showing the cytotoxic effect of T7 transcribedsiRNAs. HEK-293 cells were transfected with 20 nM of synthetic or T7transcribed siRNA and monitored microscopically on day 5 posttransfection.

FIGS. 2A-2D show interferon induction by transcribed siRNAs.

FIG. 2A is a graph showing that the anti-HSV-1 activity is mediated byinduction of interferon α and β. The anti-HSV-1 activity was assayedusing the medium from siRNA-transfected cells. Either a single (col. 3or 4) or both neutralizing antibodies (col. 5) were tested.

FIG. 2B is a graph showing anti-HSV-1 activity of T7-transcribed siRNAsin HEK-293, HeLa and CV1 cell lines. 20 nM of T7-transcribed siRNA wastransfected into the three different cell lines and HSV-1 infection wasmonitored.

FIG. 2C are graphs showing that RNAi and interferon induction areindependent phenomena. Two different siRNAs, one targeting a susceptiblesite and the other a nonsusceptible site in EGFP, were synthesizedchemically or transcribed by T7RNA polymerase and tested for RNAiefficacy (top) and interferon induction (bottom). The RNAi assayrepresents the average of three independent assays. The interferonresults are the average of two independent experiments.

FIG. 2D is a graph showing the potency of the RNA-mediated anti-HSV-1activity. The inhibition of HSV-1 infection was tested aftertransfection using the indicated amounts of synthetic or T7-transcribedsiRNAs. The average of two independent experiments is presented.

FIG. 3A are photographs showing the anti-EMCV activities of T7transcribed siRNAs, produced in accordance with the present invention,compared with the anti-EMCV activities of Poly IC. Top panel, cells;Second panel from top, Cells infected with EMCV; Third panel from top,T7 siRNA transfected cells infected with EMCV (triphosphate containinganti-EMCV T7 siRNAs stimulate interferon, thus, protecting cells fromEMCV infection); Bottom panel, Poly IC transfected cells infected withEMCV (poly IC is toxic and cells are expressing EGFP, so toxicityresults in cell death and loss of EGFP signal.)

FIG. 3B are photographs showing anti-EMCV activities of T7 transcribedsiRNAs, produced in accordance with the present invention, compared withthe anti-EMCV activities of various endoribonuclease prepared siRNAs(EsiRNA I, II, and III).

FIG. 3C are photographs showing anti-EMCV3 activities of T7 transcribedsiRNAs, produced in accordance with the present invention. Top panel,irrelevant T7 siRNA; Middle panel, EMCV3 T7 siRNA; Bottom panel, EMCV3T7 siRNA in the presence of CIP.

FIG. 4 is a schematic showing a T7 siRNA having a 5′-triphosphateproduced in accordance with the present invention. The schematic showsthe non-base paired nucleotide (G) to which the 5′triphosphate isattached. The schematic also shows the synergistic effects of the5′-triphosphate mediated innate immune response and the siRNA mediatedRNAi effect.

FIG. 5A is a graph showing the synergistic effect of siRNAs andtriphosphates in protecting cells from cytopathic effects of EMCVinfection at a MOI of 3. T7 siRNA is an siRNA molecule that has beentranscribed by T7 polymerase and that is non-target specific. EMCVsiRNA3 is an siRNA molecule that has been transcribed by T7 polymeraseand is target specific to the sequence shown in FIG. 6. EMCV siRNA3/CIPis EMCV siRNA3 that has been treated with calf intestine phophatase(CIP). The “cell only” are cells that have not been infected with EMCV.

FIG. 5B is a graph showing the synergistic effect of siRNAs andtriphosphates in protecting cells from cytopathic effects of EMCVinfection at a MOI of 10. T7 siRNA is an siRNA molecule that has beentranscribed by T7 polymerase and that is non-target specific. EMCVsiRNA3 is an siRNA molecule that has been transcribed by T7 polymeraseand is target specific to the sequence shown in FIG. 6. EMCV siRNA3/CIPis EMCV siRNA3 that has been treated with calf intestine phophatase(CIP). The “cell only” are cells that have not been infected with EMCV.

FIG. 6 is a schematic of the 5′UTR of a EMCV viral genome (SEQ ID NO:1). Also shown are the regions where siRNAs, produced in accordance withthe present invention, bind the EMCV viral genome.

FIGS. 7A-7C show the role of the initiating triphosphate in interferoninduction.

FIG. 7A shows siRNAs synthesized in accordance with the invention. (i)The EGFP #2 synthetic I (SEQ ID NOs:2 and 3), chemically synthesizedsiRNA against the EGFP #2 site, EGFP #2 synthetic II; (ii) the EGFP #2synthetic II (SEQ ID NOs:4 and 5) with 5′ OH-GGG; (iii) the EGFP #2 T7(SEQ ID NOs:4 and 5), T7 RNA polymerase-transcribed siRNA against EGFP#2 containing 5′ pppGGG; (iv) the EGFP #2 T7 (19-AA) (SEQ ID NOs:6 and7), the same as EGFP #2 T7 RNA polymerase-transcribed siRNA except forreplacing the 3′ UU with 3′ AA; (v) the EGFP #2 T7 (21-AA) (SEQ ID NOs:8and 9), T7 RNA polymerase-transcribed siRNA with 21 nt complementary tothe EGFP #2 site but including the 3′ AA. The potential cleavage sitefor RNAse T1 is boldface. The 3′ AA replacing the 3′ UU is in italics.The AA complementary to UU is underlined.

FIG. 7B are a graph and gel photograph showing triphosphate-mediatedinterferon induction [γ-³²P] GTP-labeled siRNAs were treated using eachof the conditions described below and electrophoresed in a native gel(top). RNAs (1 μg) were electrophoresed in a 15% polyacrylamide gel andstained with ethidium bromide (middle). 20 nM of siRNAs was transfectedinto HEK-293 cells and assayed for interferon β (bottom panel). Column1, the EGFP #2 T7 siRNA without T1 treatment; column 2, with T1treatment; column 3, with T1 and CIP treatment. Column 4, EGFP #2 T7(19-AA) siRNA without T1 treatment; column 5, with T1 digestion; column6, with T1 and CIP treatment.

FIG. 7C is a graph showing that T7 siRNAs (21-AA) in accordance with theinvention are effective in RNAi. HEK-293 cells were cotransfected withthe EGFP reporter plasmid and each of the siRNAs. The percentages ofEGFP expression relative to the non-siRNA-treated controls were used asthe assay for RNAi. Each value is the average of two independent assays.

FIGS. 8A-8C show induction of interferon by in vitro transcribed ssRNAs.

FIG. 8A is a graph showing that ssRNAs transcribed in vitro elicit theanti-HSV-1 effect. Mock 1: before transfection, the RNA sample was mixedwith 1 μg of RNase A. Mock 2: transfection of RNA containingtriphosphate done in the absence of a transfection reagent. T7 siRNA1and 2 are the T7 siRNAs for HSV #1 and anti-SF3A3 #1, respectively. TheT7 ssRNA is the sense RNA strand of HSV#1. The T7 EGFP wasRNA-transcribed from an EGFP-encoding DNA template. T7 (CUG)₁₃₀ is aT7-transcribed RNA harboring 130 repeats of CUG. All RNAs were used at aconcentration of 20 nM.

FIG. 8B is a graph showing the anti-HSV-1 activities of ssRNAstranscribed from T7, T3 and Sp6 polymerases. The templates of T3 ssRNA 1and 2 were created from the pBluecript II SK vector digested with EcoRIand BamHI, respectively. The templates of SP6 ssRNA 1 and 2 were createdby the EcoRI and SalI digestion of the pGEM 9Df(−) vector. The T7 ssRNAis the sense sequence of HSV #1.

FIG. 8C is a graph and gel photographs showing that the 5′ triphosphateof the transcribed ssRNA is essential for the induction of interferon.The EGFP RNA was transcribed in the presence of [γ-32P]GTP andtransfected into cells without any further modification (col. 2 and 3),after gel purification (col. 4 and 5), and after gel purification andCIP treatment (col. 6 and 7). The induced levels of interferon β weredetermined by an ELISA (top). Transcribed RNAs used for transfectionreactions were analyzed in a nondenaturing agarose gel (middle). Removalof the triphosphate by CIP was monitored on the bottom gel. The ELISAdeterminations represent the average of two independent experiments.

FIG. 9 is a graph showing induction of interferon α in 4 mice sampleswhich were injected with 70 uM triphosphate T7 siRNAs produced inaccordance with the present invention. Interferon α induction is shownin mice using mouse ELISA kit at day 1, day 3 and day 7 followinginjection of T7 siRNA.

FIGS. 10A-10D show that the 5′ triphosphate label of RNA is a novelmotif for stimulating the innate immune response.

FIG. 10A shows total RNA that was purified from influenza viral RNA andtreated without (−CIP) or with (+CIP) calf intestinal phosphatase.

FIG. 10B shows HEK293 cells that were transfected with no RNA (mock),influenza viral RNA without CIP treatment (Flu RNA −CIP), or the RNAwith CIP treatment (Flu RNA +CIP) and sequentially challenged byEGFP-labeled HSV. The infection of virus was monitored by florescencemicroscopy.

FIG. 10C shows HEK293 cells that were transfected with influenza viralRNAs without CIP pretreatment (second column) or with pretreatment at 10(third) and 60 minutes (fourth column).

FIG. 10D shows NIH3T3 cells stably expressing EGFP that were treatedwith no RNA (mock), 1 nM of T7 RNA (T7 RNA), 0.5 ug of influenza viralRNA without CIP pre-treatment (Flu RNA-CIP), and the viral RNA with CIPpre-treatment (Flu RNA+CIP). The next day (24 hours), cells werechallenged with EMCV infection. On day 3, the viral infection mediatedcytotoxic effect was monitored under light (the first panel) orfluorescence microscopy (the second panel).

FIGS. 11A-11C shows that the nuclear derived nascent RNAs indicate thedependence of the 5′ triphosphate motif for antiviral activity.

FIG. 11A shows cytoplasmic and nuclear extracts that were prepared fromHEK293 cells and tested by Western blot for the cytoplasmic proteinenolase or nuclear protein hnRNP H.

FIG. 11B shows cytoplasmic (the first lane) and nuclear RNAs (second andthird lanes) that were purified from each extract and analyzed on a 1%agarose gel in the absence (second lane) or presence of CIPpre-treatment (third lane).

FIG. 11C shows HEK293 cells that were transfected with each indicatedRNAs and sequentially infected with EGFP-labeled HSV. The pictures weretaken under florescence microscopy on day 3.

FIGS. 12A-12C show that the T7 RNA and poly IC activate the TLR3receptor and share similar expression profiles.

FIG. 12A shows total cDNA from NIH3T3 cells transfected by the T7 RNA orpoly IC that were detected and quantitated by microarray analysis. Theexpression profiles were compared between mock treated vs. T7 RNA (thefirst column), mock vs. poly IC (the second column), and T7 RNA vs. polyIC (the third column). The bar represents the total number of geneswhere were up or down-regulated by more than three-fold using a total of16,281 elopements and an average of two independent trials.

FIG. 12B shows that TLR3 is upregulated by poly IC as well as by T7 RNA.Total RNA of NIH3T3 cells were harvested after transfection with no RNA,T7 RNA, and poly IC. Based on the microarray data in FIGS. 14A-14D, theexpression level of TLR3 was compared. TLR7 and beta-actin were used asinternal controls.

FIG. 12C shows that TLR3 is required for the T7 RNA mediated innateimmune response. MRC-5 cells were pre-incubated in the presence ofanti-TLR2 or TLR3 antibodies and incubated in the presence of theindicated RNAs. The secreted interferon beta in the media was determinedby ELISA in three independent assays.

FIGS. 13A-13B show that all 86 genes upregulated by the T7 transcribedRNA were also upregulated by poly IC.

FIGS. 14A-14D show that poly IC activated a large number of additionalgenes in comparison to genes activated by T7 transcribed RNA.

DETAILED DESCRIPTION OF THE INVENTION

The ability to detect pathogenic invasion is the first line of defensein a cell and represents the most important task of the innate immuneresponse. As shown herein, siRNAs transcribed by T7 RNA polymerasedisplay a potent anti-viral effect that is dependent on the presence ofa 5′ triphosphate motif. We suggest that the innate immune response isactivated by the recognition of this RNA motif. It is also shown hereinthat Influenza A viral RNA induces 5′ triphophate dependent anti-viralactivity through the activation of the interferon response pathway whentransfected directly into cells. Nuclear-derived RNAs which include manyuncapped small RNAs and ribosomal RNAs harboring a 5′ triphosphate labelalso activate a strong interferon induction when transfected into cells.Alkaline phosphatase treatment of these RNAs eliminates this stimulationand cytoplasmic-derived RNAs, which are largely devoid of triphosphate,also fail to induce an interferon response. The 5′ triphosphatecontaining RNAs appear to be recognized by and activateToll LikeReceptor 3 (TLR3). Microarray and functional analyses indicate that5′triphophate containing RNAs constitute a novel immunostimulatory motifwhich is highly effective at inducing IFN responses in leading to potentantiviral activity in a variety of cell lines.

An embodiment of the present invention provides a method for inducing aninterferon response in a cell comprising introducing into the cell aneffective amount of a double-stranded RNA molecule, preferably aninterfering RNA (RNAi) molecule, having a triphosphate, preferably a5′-triphosphate. The presence of the 5′-triphosphate was found to inducethe interferon response. The invention also encompasses variations ofthe triphosphate which enable induction of effective amounts ofinterferon. In a preferred embodiment, the RNAi molecule having atriphosphate, and preferably a 5′-triphosphate, induces one or more ofinterferon α and β. It is understood that the expressions “having atriphosphate” or “having a 5′-triphosphate” encompass having one or moretriphosphates or 5′-triphosphates.

In a preferred embodiment, the double-stranded RNA, and preferably RNAi,molecule having a triphosphate, preferably a 5′-triphosphate, isproduced in vitro by a phage polymerase, preferably a bacteriophage RNApolymerase. Preferably the nucleotide to which the triphosphate isattached is not base-paired to the opposite strand of thedouble-stranded molecule (FIG. 4). Preferably the RNAi molecule having a5′-triphosphate is produced in vitro by a bacteriophage T7 RNApolymerase. In further embodiments of the invention, the RNAi moleculehaving a 5′-triphosphate may be produced by other phage polymerases,including a bacteriophage T3 RNA polymerase or a bacteriophage Sp6 RNApolymerase.

In another embodiment the double-stranded RNA, preferably RNAi, moleculehaving a triphosphate is synthetic or chemically synthesized.

The RNA molecules of the invention also can be purified using acceptablemethods known in the art.

In a preferred embodiment, the RNAi molecule having a 5′-triphosphate isa small interfering RNA (siRNA). In another preferred embodiment, theRNAi molecule having a 5′-triphosphate is a short hairpin RNA (shRNA)molecule. The double-stranded RNA preferably has two triphosphates, andmost preferably two 5′-triphosphates. The double-stranded RNA,preferably RNAi, and more preferably siRNA, molecule also is preferablyabout 10 to about 25 nucleotides in length, and more preferably about 20nucleotides in length, while the shRNA, which can be used to produce apreferred siRNA, is preferably about 21 to about 29 nucleotides inlength. In particular, it was found that triphosphate-containingdouble-stranded RNA as short as 10 nucleotides induced an interferonresponse. Longer RNA molecules were found to induce interferon as well,but the total concentration of the 5′-triphosphate is reduced.Therefore, the longer the RNA molecule, the more of the molecule ispreferred, since the triphosphate is believed to effect interferoninduction.

In another embodiment, the invention provides a composition for inducingan interferon response comprising an effective amount of an RNAimolecule having a triphosphate, preferably a 5′-triphosphate, whereinthe presence of the 5′-triphosphate has been found to induce theinterferon response.

In other embodiments, the invention provides an anti-viral reagent and amethod for inducing an antiviral response, comprising introducing into acell an effective amount of an RNAi molecule having a triphosphate,preferably a 5′-triphosphate, wherein the presence of the5′-triphosphate induces an interferon response, and provides ananti-viral response. The anti-viral response can be mediated byinterferons, or alternatively by both interferons and asequence-dependent RNAi effect. The anti-viral response is not limitedto mediation by interferons, but may include other cytokines orsignaling pathways. In a preferred embodiment, the RNAi molecule havinga 5′-triphosphate can be introduced into a cell prior to viralinfection, thereby, inhibiting viral infection. The present invention isuseful against any virus, including but not limited to, herpes simplexvirus 1 (HSV-1), encephalomyocarditis virus (EMCV) or Influenza A virus.

The present invention can be practiced in vitro or in vivo. Theinvention also can be used as a therapeutic or preventative agent,preferably for therapy or prevention of a disease or condition.

An effective amount refers to that amount of RNA effective to producethe intended result, including the intended pharmacological, therapeuticor preventive result. In cell culture, an effective amount forinitiating an antiviral effect can be as low as 1 nM, and can range upto 20 nM or more. However, it is understood that higher dosages can betoxic to cells, due to unregulated induction, resulting in undesiredlevels of expression of several cytokines, including interferon. Apharmaceutically effective amount or dose is that amount or doserequired to prevent, inhibit the occurrence, or treat (alleviate asymptom to some extent, preferably all of the symptoms) of a diseasestate. The pharmaceutically effective amount or dose depends on the typeof disease, the composition use, the route of administration, the typeof mammal being treated, the physical characteristics of the specificmammal under consideration, concurrent medication, and other factorswhich those skilled in the medical arts will recognize. Generally, aneffective amount or dose of dsRNA or ssRNA for human use is known in theart and/or can be determined by standard methods, and can beadministered, for example, in the ranges of about 0.001 mg/kg to 100mg/kg body weight/day or about 0.01 mg/kg to 10 mg/kg body weight/day.

Fire, A. et al. (2003), which is incorporated herein by reference,refers to introducing RNA in an amount which delivers at least one copyper cell, as well as administering higher dosages (e.g., 5, 10, 100,500, 1000, etc., copies per cell) of double-stranded RNA to yield betterresults. Ackermann, E. J. et al. (1999), which is incorporated herein byreference, describes as follows: “The formulation of therapeuticcompositions and their subsequent administration is believed to bewithin the skill of those in the art. Dosing is dependent on severityand responsiveness of the disease state to be treated, with the courseof treatment lasting from several days to several months, or until acure is effected or a diminution of the disease state is achieved.Optimal dosing schedules can be calculated from measurements of drugaccumulation in the body of the patient. Persons of ordinary skill caneasily determine optimum dosages, dosing methodologies and repetitionrates. Optimum dosages may vary depending on the relative potency ofindividual oligonucleotides, and can generally be estimated based onEC₅₀'s found to be effective in in vitro and in vivo animal models. Ingeneral, dosage is from 0.01 μg to 100 g per kg of body weight, and maybe given once or more daily, weekly, monthly or yearly, or even onceevery 2 to 20 years. Persons of ordinary skill in the art can easilyestimate repetition rates for dosing based on measured residence timesand concentrations of the drug in bodily fluids or tissues. Followingsuccessful treatment, it may be desirable to have the patient undergomaintenance therapy to prevent the recurrence of the disease state,wherein the oligonucleotide is administered in maintenance doses,ranging from 0.01 μg to 100 g per kg of body weight, once or more daily,to once every 20 years.”

Methods for formulating compositions and reagents in accordance with theinvention, as well as modes of administration, are known in the art andare described, for example, in Agrawal, S. et al. (2003) and Ackermann,E. J. et al. (1999), which are fully incorporated herein by reference.Formulations can include a pharmaceutically or physiologicallyacceptable carrier, such as an inert diluent or an assimilable ediblecarrier. The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g. byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful. Compositions and formulationsfor oral administration include powders or granules, suspensions orsolutions in water or non-aqueous media, capsules, sachets or tablets.Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids orbinders may be desirable.

Methods for delivering the RNA molecules of the invention into cellsalso are well known in the art. See Thompson, J. et al. (2004) and Fire,A. et al. (2003), which are fully incorporated herein by reference. RNAmay be directly introduced into the cell (i.e., intracellularly); orintroduced extracellularly into a cavity, interstitial space, into thecirculation of an organism, introduced orally, or may be introduced bybathing an organism in a solution containing the RNA. Methods for oralintroduction include direct mixing of the RNA with food of the organism,as well as engineered approaches in which a species that is used as foodis engineered to express the RNA, then fed to the organism to beaffected. Physical methods of introducing nucleic acids, for example,injection directly into the cell or extracellular injection into theorganism, may also be used. Vascular or extravascular circulation, theblood or lymph system, the phloem, the roots, and the cerebrospinalfluid are sites where the RNA may be introduced.

Physical methods of introducing nucleic acids include injection of asolution containing the RNA, bombardment by particles covered by theRNA, soaking the cell or organism in a solution of the RNA, orelectroporation of cell membranes in the presence of the RNA. A viralconstruct packaged into a viral particle would accomplish both efficientintroduction of an expression construct into the cell and transcriptionof RNA encoded by the expression construct. Other methods known in theart for introducing nucleic acids to cells may be used, such aslipid-mediated carrier transport, chemical-mediated transport, such ascalcium phosphate, and the like. Thus the RNA may be introduced alongwith components that perform one or more of the following activities:enhance RNA uptake by the cell, promote annealing of the duplex strands,stabilize the annealed strands, or other-wise increase inhibition of thetarget gene.

Methods for the delivery of nucleic acid molecules also are described inAkhtar and Juliano (1992) and Akhtar (1995), each of which isincorporated herein by reference. Sullivan et al. (1994) furtherdescribes the general methods for delivery of enzymatic RNA molecules.These protocols can be utilized for the delivery of virtually anynucleic acid molecule. Nucleic acid molecules can be administered tocells by a variety of methods known to those familiar to the art,including, but not restricted to, encapsulation in liposomes, byiontophoresis, or by incorporation into other vehicles, such ashydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesivemicrospheres. Alternatively, the nucleic acid/vehicle combination islocally delivered by direct injection or by use of an infusion pump.Other routes of delivery include, but are not limited to oral (tablet orpill form), intrathecal, mucosal, or transdermal delivery. Otherapproaches include the use of various transport and carrier systems, forexample, through the use of conjugates and biodegradable polymers. Moredetailed descriptions of nucleic acid delivery and administration areprovided in Sullivan et al. (1994), Draper et al. (1993), Beigelman etal. (1999) and Klimuk et al. (1999), all of which are incorporated byreference herein.

In accordance with the present invention, interferon induction andanti-viral activity can be induced in response to a variety of RNAimolecules. To test for interferon induction and antiviral activity of anRNAi molecule, first, RNA interference was tested in one embodiment as amethod to block herpes simplex virus 1 (HSV-1) infection. To performthis test, two siRNAs targeting the early ICP4 gene transcript werecreated using T7 RNA polymerase. The sequences of these are provided inTable 1. To monitor viral infection, an HSV-1 recombinant that containsthe gene encoding VP20 fused to the gene encoding the enhanced greenfluorescent protein (EGFP) was used (Elliott, G. and O'Hare, P., 1999).When cells are infected with the virus, they express EGFP, allowingsimple microscopic assays for viral infectivity. First, human embryonickidney (HEK) 293 cells were transfected with the siRNAs (10 nM each)before viral infection. Twenty hours later the recombinant HSV-EGFPvirus was added to the cultured cells at a multiplicity of infection(MOI) of 1. Twenty-four hours after viral challenge, infectivity wasmonitored by microscopic analysis of EGFP expression. The two siRNAstargeting HSV-1 as well as one of the irrelevant siRNA controlsinhibited viral infectivity dramatically (FIG. 1A). When analyzing theresults of these experiments a correlation was found between theanti-HSV-1 activity and the source of the siRNAs. Whereas the twochemically synthesized siRNAs showed no anti-HSV-1 activity, all threeof the siRNAs prepared by in vitro transcription using T7 RNA polymeraseshowed potent HSV-1 inhibition. Next, an siRNA with a sequence identicalto the chemically synthesized control siRNA, the RNA binding protein La(Table 1), which did not have antiviral activity, was transcribed usinga T7 RNA polymerase. The T7 RNA polymerase-transcribed version of thissiRNA elicited a potent anti-HSV-1 response, supporting the hypothesisthat some component of the T7 siRNA was eliciting an anti-HSV-1 responsein a non-sequence-dependent manner (FIG. 1A).

TABLE 1 Sequence of siRNAs 5′ sequence 3′ Id of siRNAs (SEQ ID NO:)Source Anti-La#2 AACTGGATGAAGGCTGGGTAC Dharmacon (10) Anti-Ro#1AATCTGTAAACCAAATGCAGC Dharmacon (11) Anti-HSV#1 AACAAGCAGCGCCCCGGCTCC T7transcription (12) Anti-HSV#2 AACAGCAGCTCCTTCATCACC T7 transcription(13) Anti-SF3A3#1 AAGGAACGGCTCATGGACGTC T7 transcription (14)

To further investigate the nature of the T7 siRNA-mediated inhibition ofHSV-1 infection, HEK-293 cells were transfected with the chemicallysynthesized or T7-transcribed siRNAs and monitored for cell growth. TheT7 siRNA-transfected cells underwent cell death after 5 days, indicativeof possible activation of the interferon response pathway in response tothe T7 transcripts (FIG. 1B). It was also found that the anti-HSV-1effect was transferable with the medium of T7 siRNA-transfected cells,evidence of secreted protein(s), which further supports the likelihoodof an interferon-mediated response. To verify the presence of aninterferon-mediated response, the medium of T7 siRNA-transfected cellswas assayed for interferon α and β using an enzyme-linked immunosorbentassay (ELISA). Substantial amounts of both interferons were induced bytransfection of 10 nM of the T7 siRNAs (Table 2).

TABLE 2 Induction of interferon by the T7 siRNAs Amount of Interferon αAmount of Interferon β SiRNA (10 nM) (pg/ml) (pg/ml) Mock 0.2 ± 0.3 3 ±2 Synthetic siRNA   2 ± 0.5 5 ± 5 T7 siRNA 1 (anti-La #2) 300 ± 85 4,000 ± 300   T7 siRNA 2 (anti-Ro #1) 250 ± 35  3,500 ± 300  

To confirm that the anti-HSV-1 effect was mediated by the interferons,HSV-1 infection was tested using medium from T7 siRNA-transfected cellspreviously treated with neutralizing antibodies (FIG. 2 a). Acombination of antibodies to interferon α and β was required toneutralize the inhibition, suggesting that both interferons aremediating the antiviral response. In this embodiment, inhibition ofHSV-1 infection took place preferably when cells were pretreated withthe T7 siRNAs, or in another preferred embodiment when medium from theT7 siRNA-treated cells was added to a fresh cell culture before HSV-1challenge. These results are consistent with the known mechanisms ofinterferon inhibition of HSV-1 and the anti-interferon mechanism of thisvirus, which shuts down the host response during infection (Samuel, C.E., 2001). In this embodiment, the interferons were induced beforeinfection, presumably triggering the expression of antiviral genes(Samuel, C. E., 2001).

Because the initial experiments in the above embodiment were done withHEK-293 cells, and at least one report shows that HEK-293 cells lack anantiviral interferon response (Stojdl, D. F., et al., 2000), other celllines were tested for their T7 siRNA-mediated interferon response. Forexample, both HeLa cells and African Green Monkey kidney fibroblasts(CV1) were transfected with either chemically synthesized or T7transcribed-siRNAs (FIG. 2 b). In each case the T7 transcripts eliciteda non-sequence-dependent inhibition of HSV-1, whereas the chemicallysynthesized siRNAs did not. Next, a different batch of HeLa cellsobtained from the ATCC were tested and a similar level of interferoninduction was found. In other embodiments, T7 siRNA-mediated interferonlevels were measured in media from K562, CEM and Jurkat cellstransfected with T7 siRNAs, and again interferon induction was observedin each of these media.

Further, siRNAs targeting EGFP itself were chemically synthesized ortranscribed in vitro by T7 RNA polymerase (FIG. 2C). Two different sitesin EGFP were chosen, one that is highly susceptible to siRNA knockdown,and one that is not (Kim, D. H. and Rossi, J. J., 2003). When EGFPexpression was monitored, the T7 siRNAs showed more potent RNAi than thesynthetic siRNAs (FIG. 2C, top, col. 2 versus 4, 3 versus 5). Each ofthe T7 siRNAs also showed potent interferon induction, indicating thatthe potency of the knockdown and the interferon induction are twoindependent phenomena. Unlike the T7 siRNAs, chemically synthesizedsiRNAs did not induce interferon (FIG. 2C, bottom, col. 2, 3). Inaddition to the EGFP analyses, the anti-HSV-1 activities of thechemically synthesized versus the T7-transcribed siRNAs were assayed. Upto 200 nM synthetic siRNAs did not inhibit HSV-1, whereas T7-transcribedRNAs completely inhibited HSV-1 at approximately 5 nM (FIG. 2D).

To test for interferon induction and antiviral activity of an RNAimolecule, additional tests were preformed in connection with otherembodiments using RNAi molecules and other viruses, for example,encephalomyocarditis virus (EMCV). An anti-EMCV siRNA having a5′-triphosphate, which was produced by a bacteriophage T7 RNApolymerase, was created and introduced into cells.Polyinosinic-polycylidylic acid (Poly IC) was also introduced intocells. EMCV was added to the cultured cells at a multiplicity ofinfection (MOI) of 10 and the results are shown in FIG. 3A. Triphosphatecontaining anti-EMCV T7 siRNAs stimulate interferon, thus protectingcells from EMCV infection (FIG. 3A, third panel from top). In contrast,poly IC is toxic, as seen in FIG. 3A, bottom panel. Cells are expressingEGFP, so toxicity results in cell death and loss of EGFP signal.Additional experimental results obtained under various conditions alsoare shown in FIGS. 3B and 3C.

In other embodiments, (HEK) 293, HeLa and 3T3 cells were exposed to T7siRNAs, made in accordance with the present invention. (HEK) 293, HeLaand 3T3 cells were also exposed to Poly IC. Cell type specific Poly ICand T7 triphosphate siRNA interferon responses in the different cellsunder various conditions are shown in Table 3 below.

TABLE 3 Cell type specific Poly IC and T7 triphosphate siRNA responsesInterferon beta Transfected RNA Amount Toxicity on day 2 (pg/ml) 293Mock − 0 T7 siRNA 10 nM − 300 ± 50 Poly IC 50 ng −  10 ± 10 Poly IC 100ng  + 10 ± 5 T7 siRNA + Poly IC 10 nM + 50 ng − 20 ± 5 HeLa Mock − 0 T7siRNA 10 nM −  250 ± 100 Poly IC 50 ng − 0 Poly IC 100 ng  +  20 ± 10 T7siRNA + Poly IC 10 nM + 50 ng − 0 3T3 Mock − 0 T7 siRNA 10 nM − 300 ± 75Poly IC 250 ng  − 250 ± 50 Poly IC 500 ng  + 200 ± 50 T7 siRNA + Poly IC100 nM + 250 ng − 350 ± 75

Table 3 shows the advantages of T7 triphosphate siRNAs over Poly IC inthe three different cell types. For example, T7 triphosphate siRNAs areless toxic than Poly IC. They also exhibit a more potent induction ofinterferon β and a strong antiviral effect.

In another embodiment, the invention provides a method for inducing ananti-viral response in a cell comprising introducing into a cell,preferably a mammalian cell, an RNAi molecule having a triphosphate,preferably a 5′-triphosphate, wherein the RNAi molecule induces asynergistic effect resulting from an RNAi molecule-mediated RNAi effecttogether with a 5′-triphosphate-mediated interferon response.

In another embodiment, the invention provides an anti-viral reagentcomprising an RNAi molecule having a triphosphate, preferably a5′-triphosphate, wherein the RNAi molecule induces both an RNAi effectand an interferon response.

In a preferred embodiment, the RNAi molecule having a 5′-triphosphate,can be produced in vitro by a bacteriophage T7 RNA polymerase. In otherembodiments of the invention, the RNAi molecule having a 5′-triphosphatecan be produced by other phage polymerases, including but not limitedto, a bacteriophage T3 RNA polymerase and a bacteriophage Sp6 RNApolymerase. In another embodiment, the RNAi molecule having a5′-triphosphate is chemically synthesized.

In preferred embodiments, the RNAi molecule having a 5′-triphosphate isa siRNA or a shRNA molecule. The RNAi molecule, or other double-strandedRNA molecule, can be those otherwise known in the art.

In the above embodiment, when an RNAi molecule having a 5′-triphosphate,preferably a short dsRNA and more preferably an siRNA, is designed in asequence specific manner, potent anti-viral effects can be detected asthe result of synergistic effects of the 5′-triphosphate mediated innateimmune response, i.e., interferon mediated response, of cells and thesiRNA mediated RNAi effect (FIG. 4). Since this anti-viral effect ismuch more than a simple additive effect of an interferon response andsiRNA mediated RNAi, the RNAi molecule can be a powerful anti-viralreagent.

In a preferred embodiment, FIG. 5A shows the synergistic effect ofsiRNAs and triphosphates in protecting cells from the cytopathic effectsof EMCV infection at a MOI of 3. In another embodiment, FIG. 5B showsthe synergistic effect of siRNAs and triphosphates in protecting cellsfrom the cytopathic effects of EMCV infection at a MOI of 10.

Another embodiment of the present invention provides a method forinducing an interferon response in a cell, comprising introducing intothe cell, preferably a mammalian cell, a single stranded RNA (ssRNA)having a triphosphate, preferably a 5′-triphosphate, wherein thepresence of the 5′-triphosphate induces the interferon response. In apreferred embodiment, the ssRNA having a 5′-triphosphate induces one ormore of interferon α and β.

In a preferred embodiment, the ssRNA having a 5′-triphosphate isproduced in vitro by a bacteriophage RNA polymerase. More preferably thessRNA having a 5′-triphosphate is produced in vitro by a bacteriophageT7 RNA polymerase. In other embodiments, the ssRNA having a5′-triphosphate may be produced by other phage polymerases, includingbut not limited to, a bacteriophage T3 RNA polymerase and abacteriophage Sp6 RNA polymerase.

In another embodiment, the ssRNA having a triphosphate can be chemicallysynthesized.

The preferred lengths of short ssRNAs having a triphosphate, preferablya 5′-triphosphate, are expected to be roughly the same as fordouble-stranded RNA molecules. At least one difference is that theeffect of length of a ssRNA molecule may vary depending on the celltype. For example, while some cells show an effect similar for dsRNAs,others may not as a result of being mediated by different nucleaseactivity. In particular, similar effects can occur in both ssRNA anddsRNA in certain cell lines, such as HEK 293 cells, while in other celllines lesser of an effect may be observed with ssRNA on account of ssRNAbeing less stable than dsRNA.

In another embodiment, an antiviral response can be induced byintroducing into a cell an ssRNA having a triphosphate, preferably a5′-triphosphate, wherein the presence of the 5′-triphosphate induces aninterferon response as well as an anti-viral response. In anotherembodiment, the ssRNA is introduced into a cell prior to viralinfection, and thereby inhibiting viral infection. Viruses include, forexample, herpes simplex virus 1 (HSV-1), EMCV or Influenza virus A, aswell as other viruses.

To confirm the role of the triphosphate, T7 RNA polymerase-transcribedssRNAs were tested as well (FIG. 8A). No HSV-1 inhibition was observedwhen cells were transfected with the RNase A-treated ssRNAs (FIG. 8A,mock 1) or T7 ssRNA in the absence of a transfection reagent (FIG. 8A,mock 2), but anti-HSV-1 activity was observed when cells weretransfected with a non-RNAse-treated-ssRNA in the presence of cationiclipid. Additional experiments were carried out in other embodimentsusing ssRNAs transcribed by the bacteriophage T3 and Sp6 RNA polymerases(FIG. 8B). Each of these transcripts also elicited anti-HSV-1 activity.

Interferon assays from these experiments indicate that the ssRNAs arealso potent inducers of interferons (Table 4).

TABLE 4 Induction of interferon mediated by various in vitro transcribedRNAs. Amount of Interferon-α Amount of Interferon-β RNAs (pg/ml) (pg/ml)T7 siRNA¹ 10 nM 300 ± 85  4,000 ± 300 40 nM 650 ± 100 9,500 + 500 T7single stranded RNA² 10 nM 580 ± 120 8,000 ± 250 40 nM 1050 ± 280 10,000 ± 500  T3 single stranded RNA³ 10 nM 620 ± 180 7,000 ± 500 40 nM1000 ± 240  10,000 ± 1000 Sp6 single stranded RNA⁴ 10 nM 600 ± 50  7,000± 500 40 nM 800 ± 80  8,000 ± 300 ¹Anti-La #2 siRNA. ²The sense RNA ofHSV #1. ³The T3 transcript from the BamHI digested pBluescript DNAtemplate. ⁴The Sp6 transcript from the SalI digested pGEM9Df(−) DNAtemplate.

The ssRNAs were transcribed in the presence of [γ-³²P]GTP and analyzedby gel electrophoresis confirming their single stranded nature (FIG. 8C,middle and bottom). These ssRNAs all induce interferon, but thiscapacity is lost when these RNAs are treated with calf intestinephosphatase (CIP) (FIG. 8C).

In another embodiment, the present invention provides a method forinhibiting the interferon inducing activity of a RNAi molecule having a5′-triphosphate comprising removing, preferably by cleaving, the5′-triphosphate and/or the initiating 5′ nucleotides, from the RNAimolecule, wherein removal of the 5′-triphosphate and/or nucleotidesreduces the interferon inducing activity of the RNAi molecule whilestill maintaining partial or full efficacy.

In a preferred embodiment, means are incorporated at the 3′ terminus ofthe RNAi molecule to prevent base pairing with the initiating 5′nucleotides, preferably 5′ guanines, of the molecule. In one embodiment,at least two bases, preferably one or more adenosines, are incorporatedat the 3′ terminus of the RNAi molecule to prevent base pairing with oneor more initiating 5′ guanines of the RNAi molecule prior to cleavingthe 5′-triphosphate and/or nucleotides from the RNAi molecule.Incorporation of the bases thereby allows the cleavage means, preferablya ribonuclease and/or phosphatase, to remove the initiating5′-triphosphates and/or nucleotides of the transcripts.

In another preferred embodiment, the invention provides a method forinhibiting interferon inducing activity of a ssRNA having a5′-triphosphate comprising removing, preferably by cleaving, the5′-triphosphate and/or initiating 5′ nucleotides from the ssRNA, whereinremoval of the 5′-triphosphate and/or nucleotides reduces the interferoninducing activity of the ssRNA.

The cleavage step is performed preferably by a nuclease, more preferablya ribonuclease, preferably T1 ribonuclease, or by a phosphatase,preferably calf intestine phosphatase (CIP). However, it is understoodthat other means and enzymes can be used to effect the cleavage.

In a preferred embodiment, the cleavage step is performed by both aribonuclease and a phosphatase, preferably T1 ribonuclease and calfintestine phosphatase (CIP).

To determine the active interferon-inducing agent in the RNAi molecules,a series of experiments were carried out focusing on the initiating Gresidues. Because T7 RNA polymerase initiates transcription with5′-pGGG, a determination was made as to whether the GGG associated withthe 5′ end of the transcript was the inducing agent by chemicallysynthesizing the anti-EGFP #2 siRNA (FIG. 7A, EGFP #2 synthetic 1) witha 5′-OH-GGG, and testing this siRNA for interferon induction. Nointerferon induction in HEK-293 cells was elicited by this siRNA.

The other major difference between the synthetic and in vitroT7-transcribed siRNAs is the 5′ triphosphate. The anti-EGFP #2 siRNA wastranscribed by T7 RNA polymerase in the presence of [γ-³²P]GTP to labelthe γ-phosphate. The initiating pGGG should be cleaved from thetranscript by the single strand-specific ribonuclease T1 (Wang, L., etal., 1976) if the Gs are within a single-stranded region of the siRNA.When the anti-EGFP #2 siRNA was treated with RNase T1, there was amodest reduction in interferon induction compared with the untreatedsiRNA (FIG. 7B, bottom, col. 2). When the RNA was sequentially createdwith ribonuclease T1 and calf intestine phosphatase (CIP), theinterferon induction was further reduced (FIG. 7B, col. 3). For each ofthese samples, removal of the labeled 5′γ-phosphate was monitored usingnative gel electrophoresis (FIG. 7B, top). From these analyses it wasconcluded that the residual amount of siRNA containing 5′γ-triphosphatewas proportional to the extent of interferon induction.

Given that the ribonuclease T1 treatment of the anti-EGFP #2 siRNA didnot completely remove the 5′-pGGG, it was reasoned that perhaps it orthe adjacent Gs were involved in wobble base pairing with the 3′terminal Us of the transcript, making this a poor substrate for thesingle strand-specific ribonuclease and CIP. To test this possibility, aversion of the EGFP #2 siRNA that contained 19 bases complementary toEGFP followed by AA at the 3′ end (FIG. 7A, EGFP #2 T7 (19-AA) wastranscribed. When this siRNA was treated with T1 and tested in cellculture for interferon induction, there was a reduction relative to thepGGG-containing control (FIG. 7B, col. 5). When this RNA was furthertreated with CIP, the 5′ triphosphate was completely removed along withcomplete loss of interferon induction (FIG. 7B, col. 6) even at aconcentration of 100 nM. Combining these results, it was concluded thatthe interferon induction observed with the in vitro transcribed siRNAsis linked to the presence of a 5′ triphosphate.

The EGFP #2 T7 (19-AA) siRNA was also tested for EGFP knockdownactivity, but it showed little potency (FIG. 7C, col. 4). It wasreasoned that because this siRNA now contained a total of only 19 basescomplementary to EGFP, it was not as potent as an siRNA with 21complementary bases. To test this, an siRNA with 21 bases complementaryto the same EGFP target and still maintaining the two adenosines at the3′ terminus was created (FIG. 7A, EGFP #2 T7 (21-AA)). This particularsiRNA elicited a potent EGFP knockdown (FIG. 7C, col. 5) in the completeabsence of an interferon response. Thus, by preventing the formation ofbase pairs with the initiating Gs, a combination of T1 ribonuclease andCIP treatment completely eliminated the interferon response, whilemaintaining active RNAi function for these siRNAs.

Moreover, the fact that transcripts containing triphosphates are suchpotent inducers of interferon makes it of great interest to understandwhich, if any, of the interferon-linked receptors respond to thetriphosphate-containing siRNAs. The triphosphate at the 5′-end of theuncapped negative (genomic) strands of RNA viruses like influenza virus(Honda, et al. 1998) may correspond to the biological substrate targetedby the interferon response seen with T7 ssRNA. To this extent, it isimportant to understand the biological role that triphosphate inductionof interferon plays in normal cellular viral defense mechanisms.

Preferred embodiments of the invention provide an siRNA synthesized fromthe T7 RNA polymerase system, which can trigger a potent induction ofinterferon α and β in a variety of cell lines. In addition, very potentinduction of interferon α and β by short single-stranded RNAs (ssRNAs)transcribed with T3, T7 and Sp6 RNA polymerases was also found. Analysesof the potential mediators of this response revealed that the initiating5′ triphosphate is required for interferon induction.

In another embodiment, the present invention provides for short dsRNAshaving triphosphates, preferably 5′ triphosphates, which are potentenhancers of interferons as well as potential anti-viral reagents. Thepresent invention also provides for the use of short dsRNAs or RNAimolecules, e.g., siRNA or shRNA, transcribed in vitro, and not processedto remove the initiating 5′-triphosphate, which exhibit potentinterferon stimulation both in cell culture and in mice. This interferonstimulation may inhibit viral infection if the treatment is providedprior to viral infection, or in some instances, when it is providedafter viral infection. Viral infection otherwise is treatable with thepresent invention.

The present invention answers the question of how thetriphosphate-containing siRNAs and ssRNAs induce interferon. Forexample, when HEK-293 cells were transfected with up to 20 μg totalcellular RNA, no interferon induction was observed. In contrast, aslittle as 1 nM of the in vitro transcribed siRNA initiates theinterferon response (FIG. 2D). The antiviral activities of interferonsare well studied (Samuel, C. E., 2001), but the present invention isbelieved to be first to show that the presence of a triphosphate on invitro transcribed-RNAs can potently induce interferon α and β, andfurthermore elicit a strong, non-sequence-specific antiviral response toviral challenges, such as HSV-1 challenge. In contrast, other reports inwhich T7-transcribed siRNAs were used as antiviral agents did notincorporate interferon assays in the analyses (Capodici, J., et al.,2002; Kapadia, S. B., et al., 2003), and thus whether any anti-viraleffect was due to interferon induction rather than the RNAi effect wasnot recognized.

The above shows that short interfering RNAs (siRNAs) prepared by invitro transcription using T7 RNA polymerase induce potent anti-HerpesSimplex Virus 1 (HSV1) activity that is mediated by the induction oftype 1 interferons. The anti-viral activity is dependent on the presenceof a 5′ triphosphate motif on either strand of the siRNA duplex and theantiviral effects are reversed by simple treatment with calf intestinalphosphatase (CIP). We hypothesized that a host defense system existswhich recognizes 5′ triphophate-containing viral RNAs. To furthercharacterize the anti-viral properties of the 5′ triphosphate motifexperiments were performed with genomic RNA derived from the Influenza Avirus. Influenza viral RNAs lack 5′ modifications since thevirus-derived transcriptase is unable to modify the 5′ terminus of mRNAsin the cytoplasm (Lamb, R. A. and Choppin, P. A., 1983). Purifiedinfluenza viral RNAs were incubated in the presence or absence of CIPprior to transfection into HEK293 cells (FIG. 10A). The cells weresequentially challenged by HSV1 harboring an EGFP reporter gene(Elliott, G. and O'Hare, P., 1999). When cells were pre-transfected withinfluenza viral RNA, they were protected from HSV1 infection in a mannerthat was dependent on pre-treatment with CIP (FIG. 10B). The CIPtreatment is limited to the removal of the 5′ triphosphate and does notaffect the integrity of the RNA (FIG. 10A) (Kim, D. H. et al., 2004).

We further investigated if the anti-viral effect is mediated by type 1interferon induction. The level of interferon α was determined by ELISA(FIG. 10C). Consistent with the results from the anti-HSV response, theinduction of interferon α is dependent on CIP treatment, which was shownto be augmented by prolonging the exposure to CIP. To generalize thisobservation using a different model, the mouse cell line NIH3T3 stablyexpressing EGFP (Kim, D. H. et al., 2005) was used. When this cell linewas infected with Encephalomyocarditis virus (EMCV), the cytotoxiceffect of the virus was measured by the loss of EGFP expression (FIG.10D, the first vs. second row). The cytotoxic effect by EMCV was reducedwhen the cells were transfected with either T7 RNA or Influenza viralRNA prior to viral challenge (FIG. 10D, third and fourth rows). Clearlythe antiviral activity is dependent on the presence of a 5′ triphosphatemotif on introduced RNA (FIG. 10D, fourth vs. fifth rows) and thisproperty is not limited to human cells.

Since a 5′ triphosphate group present on any RNA induces an innateimmune response, there is the distinct possibility that endogenouscellular RNAs can be potentially immunogenic. Although all nascenttranscripts in the nucleus may harbor a 5′ triphosphate, it is cappedprior to cytoplasmic export (Wei, C. and Moss, B., 1977; Gu, M. andLima, C. D., 2005). To test whether endogenous cellular RNAs fromdifferent compartments can elicit an immune response, cytoplasmic andnuclear extracts of HEK293 cells were prepared. The integrity of thefractionate samples was confirmed by Western blot analyses to detect thenuclear specific hnRNP H (Chou, M. Y., et al., 1999) or cytoplasmicspecific enolase (Dolken, G. et al., 1975) (FIG. 11A). The majority ofenolase staining was in the cytoplasmic fraction whereas the hnRNP Hdetection took place only in the nuclear fraction. RNA was purified fromeach fraction and used in cationic lipid mediated transfections ofHEK293 cells (FIG. 1B) which were subsequently challenged with HSV1(FIG. 11C). RNA derived from the cytoplasmic fraction did not elicit ananti-HSV protective response whereas the nuclear-derived RNAs showed ananti-viral response that could be abrogated by prior treatment with CIP(FIG. 11C). These data indicate that two types of RNAs separated by thenuclear membrane have different immunogenic characteristics mediated bythe 5′ triphosphate. These results suggest that cells have adopted theirantiviral defense strategy based on the biological principal that mostif not all cytoplasmic RNAs lack an exposed 5′ triphosphate as aconsequence of capping. Thus 5′-triphosphate-containing RNAs may berecognized as infectious viral RNAs, thereby activating the innateimmune system as a defense mechanism.

A comprehensive profile of gene expression by double-stranded RNA hasbeen previously undertaken (Geis, G. et al., 2001). However, tocharacterize the signaling pathways elicited by 5′-triphosphate labeledRNA, NIH3T3 cells were transfected with either poly IC or bacteriophageT7-generated RNA (T7 RNA initiates with a tri-phosphate) and theirrelative gene expression profiles were compared using a murineoligonucleotide microarray. RNA was extracted at three different timepoints: 4, 8, and 16 hours following transfection with T7 RNA. Unlikethe early response induced by Poly IC (Geis, G. et al., 2001), noexpression changes were detected until 16 hours post transfection,indicating that the tri-phosphate RNA mediated response takes place moreslowly than the Poly IC induced response (data not presented). T7transcribed RNA resulted in upregulation of the expression of 86 genesamong the 16,261 genes on the array when a three-fold threshold was used(FIG. 12A). In a parallel experiment, 229 genes were up-regulated, 12genes down-regulated in poly IC-transfected cells. Interestingly, all 86genes upregulated by the T7 transcribed RNA were also upregulated bypoly IC (FIGS. 13A-13B), although poly IC activated a large number ofadditional genes (FIGS. 14A-14D).

To minimize the possibility that poly IC we used in the microarray iscontaminated with impure materials such as LPS, additional microarrayexperiments were performed using purified poly IC or poly ICsupplemented with 20 ug/ml polymyxin B, which is a well-characterizedLPS inhibitor (Kariko, K., et al., 2004). The identical set of genes wasfound to be activated under all these conditions (data not presented).Among the genes whose expression was induced by poly IC, several genesrelated to the apoptosis pathway have been identified as previouslyreported (FIGS. 14A-14D) (Der, S. D. et al., 1997). When the T7transcribed RNAs was treated with CIP prior to transfection, theexpression profile was identical to that of the non-transfected controlcells (data not presented). We confirmed some of the induced geneexpression identified in the microarray analyses using quantitativeRT-PCR for each representative group of genes. Expression of two genesupregulated in cells transfected with both T7 RNA and Poly IC (Ifi44 andTgtp) or upregulated when only transfected with Poly IC (Tnf3ip3 andGadd4alpha) were tested with an internal control (beta-actin) wasconfirmed in this manner (data not presented).

The Toll like receptors respond to pathogens that present certainmotifs, termed the pathogen-associated molecular pattern (PAMP), thatare displayed on the surface of the invading organisms (Beutler, B., etal., 2004a; Boehme, K. W. and Compton, T., 2004; Beutler, B., 2004b). Todefine the receptor for the T7 transcribed, tri-phosphate containingRNA, the expression of Toll Like Receptors were compared in microarraydata generated from RNA-transfected NIH3T3 cells. The microarray resultsshow that the T7 transcribed RNA induces TLR3 expression (data notpresented) which was further confirmed by RT-PCR. TLR3 expression wasdetermined by RT-PCR in poly IC-treated cells, which is known to possessa PAMP for TLR3 (Alexopoulou, L., et al., 2001) (FIG. 12B). Recognitionof T7 transcribed RNAs by TLR3 was additionally confirmed by afunctional inhibition assay using appropriate antibodies. IFN-βproduction of poly IC is known to be inhibited by an anti-TLR3 mAb in ahuman lung fibroblast cell line, MRC-5, which expresses TLR3 on the cellsurface (Matsumoto, M. et al., 2002). When T7 transcribed RNA orinfluenza viral RNA was transfected into these cells, expression levelsincreased similarly to Poly IC-treated cells (FIG. 12C). Interferon βinduction was inhibited when the cells were pre-incubated in thepresence of an anti-TLR3 antibody, but not by an anti-TLR2 antibody.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, immunology, cell biology, cellculture and transgenic biology, which are within the skill of the art.See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989,Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rdEd. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.);Ausubel et al., 1992), Current Protocols in Molecular Biology (JohnWiley & Sons, including periodic updates); Glover, 1985, DNA Cloning(IRL Press, Oxford); Anand, 1992; Guthrie and Fink, 1991; Harlow andLane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Jakoby and Pastan, 1979; Nucleic AcidHybridization (B. D. Hames and S. J. Higgins eds. 1984); TranscriptionAnd Translation (B. D. Hames and S. J. Higgins eds. 1984); Culture OfAnimal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); ImmobilizedCells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide ToMolecular Cloning (1984); the treatise, Methods In Enzymology (AcademicPress, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H.Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory);Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.),Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker,eds., Academic Press, London, 1987); Handbook Of ExperimentalImmunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986);Riott, Essential Immunology, 6th Edition, Blackwell ScientificPublications, Oxford, 1988; Hogan et al., Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986);Westerfield, M., The zebrafish book. A guide for the laboratory use ofzebrafish (Danio rerio), (4th Ed., Univ. of Oregon Press, Eugene, 2000).

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.Examples 1-5 relate to studies with HSV-1 and ECMV. Examples 6-12 relateto studies with Influenza A virus.

EXAMPLE 1 RNAs

The chemically synthesized RNAs were purchased from Dharmacon. The T7siRNAs were synthesized using the Silencer siRNA construction kit fromAmbion according to the manufacturer's protocol. To transcribe RNA invitro, T7 primer I (5′-TAATACGACTCACTAT A-3′ (SEQ ID NO: 15)) washybridized with T7 primer II, which contains antisense sequence of eachtranscribed RNA and the tail sequence of 5′-CCCTATAGTGAGTCGTA-3′ (SEQ IDNO:16). To make siRNA without interferon induction, the first AA wasreplaced by TT and included in the T7 primer II. For example, to makeGFP #2 T7 (21-AA), two primers were used(5′-TTAAGCTGACCCTGAAGTTCATCCCCTATAGTGAGTCGTA-3′ (SEQ ID NO:17) and5′-TTGATGAACTTCAGGGTCAGCTTCCCTATAGTGAGTCGTA-3′ (SEQ ID NO: 18)). For theCIP, 20 U of RNAs (NEB) was added to the siRNA after DNase and RNase T1digestion, and further incubated at 37° C. for 1 h. Final siRNAs werecolumn-purified using conditions recommended for the Silencer siRNAconstruction kit.

To synthesize the [γ-³²P]GTP-labeled siRNA, the transcription was donein the presence of 10 mM of cold ATP, CTP and UTP, 2 mM of GTP and[γ-32P]GTP (10 mCi/ml; ICN), and purified using a G50 column (Amersham).For RNAse T1 treatment, the RNA was incubated in the presence of 5 U ofRNase T1 in 1× buffer (50 mM Tris-HCl, pH 7.0; 5 mM EDTA; 50 mM NaCl).CIP treatment of EGFP RNA was carried out at 37° C. for 1 h at 1× buffer(100 mM NaCl, 50 mM Tri-HCl, 10 mM MgCl2, 1 mM dithiothreitol (DTT)).

For T3 RNAs, 1 μg of pBluescript DNA template (Stratagen) was digestedwith either BamHI or EcoRI and used as a template of in vitrotranscription using the T3 RNA polymerase (Promega). The Sp6 RNAs weretranscribed from the same amount of SalI- or EcoRI-digested pGEM9Df(−)DNA template using the Sp6 RNA polymerase (Promega). All transcriptionreactions were done under standard reaction conditions and contained 1μg of linearized DNA template, 2 μl of 100 mM DTT, 2 μl of 10× reactionbuffer supplied by each manufacturer, 8 μl of final 2 mM of NTP and 1 μlof each enzyme at 37° C. for 1.5 h. All RNAs were digested with 4U ofRNase-free DNAse (Ambion) for 1 h and used in the transfection assay.

EXAMPLE 2 Transfection and RNAi Assay

All transfection assays were done using Lipofectamine 2000 following themanufacturer's protocol (Invitrogen). HEK-293 cells at ninety percentconfluency were transfected in 24-well plates with the reporter genesand siRNAs. 250 ng of the pLEGFP-C1 vector (Clontech) and 10 nM of eachanti-EGFP siRNA were cotransfected. EGFP expression levels weredetermined 24 h later from the mean number of EGFP-fluorescent cellsdetermined by fluorescence-activated cell sorting (FACS) analyses.Percentages of EGFP expression were determined relative to nonspecificcontrols.

For monitoring of cell death, the medium from transfected cells waschanged 24 h after transfection. Additional media changes were madeafter 48 h, and the plates were examined microscopically after another48 h incubation.

EXAMPLE 3 Anti-HSV-1 Assays

60% confluent HEK-293 cells were transfected in 24-well dishes withsiRNA or ssRNA using Lipofectamine 2000 (Invitrogen) according to themanufacturer's protocol. The cells were placed in fresh medium 18 hafter transfection. The cells were infected 6 h later with HSV-1expressing EGFP at an MOI of 1. Cells were subject to FACS analyses 24 hafter infection to determine levels of EGFP expression.

EXAMPLE 4 Assays for Interferon α and β

The amount of interferon α and β secreted into the growth medium wasdetermined using interferon ELISA kits (RDI). The medium fromLipofectamine-complexed, RNA-transfected HEK-293 cells was collected 24h after the initial infection. The medium was serially diluted andassayed for the amount of secreted interferon according to themanufacturer's protocol. Each assay was carried out in triplicate. Theantibody neutralization assays were carried out as follows. Neutralizingantibodies for interferon α and β were purchased from RDI. The mediumwas collected 24 h after transfection with 40 nM of T7 ssRNA. The mediumwas next diluted 3.3% with the fresh medium and mixed with 100 U/ml ofone or both interferon neutralizing antibodies for 1 h. Theantibody-treated medium was added to the cell cultures and left for 24 hbefore HSV-1 challenge.

EXAMPLE 5 Interferon Assay in Mice

An assay for interferon can be performed to detect interferon inductionby triphosphate siRNAs, produced in accordance with the presentinvention, in mice. The effect of T7 siRNA in 4 mice samples wasobserved by measuring interferon α induction at day 1, day 3 and day 7following injection of T7 siRNA. (FIG. 9). Assays were performed asfollows: (1) Inject mouse with saline (mock) or 70 μM triphosphatesiRNAs minus triphosphate or 70 μM triphosphate siRNAs into leg musclein 25 μl volume; (2) Bleed the mouse days 1, 3, or 7 following RNAinjection; (3) Assay IFN a using mouse ELISA kit.

EXAMPLE 6 Materials

Reagents

Poly IC and Polymysin B were purchased from Sigma. Poly IC was furtherpurified through extraction twice with phenol followed by ethanolprecipitation. For determination of interferon alpha and beta, ELISAkits were purchased from RDI (Concord, Mass.). HEK293, NIH3T3, and MRC5cells were cultured in DMEM media supplemented with 10% Fetus BovineSerum and glutamine. The enolase antibody was purchased from Biogenesis(Kingston, HN). HnRNP H antibody was a generous gift from Dr. Black Lab(UCLA, CA). Cytoplasmic and nucleus extracts were prepared as describedwith a modification (Robb, G. G., et al., 2005). The isolated nuclei andcytoplasmic extract were mixed with Stat 60 (Tel-Test) followed by theManufacturer's instructions to purify RNA.

siRNAs

The anti-poliovirus siRNA (siC (Gitlin, L., et al., 2002); sensesequence 5′-GCGUGU AAUGACUUCAGCGUG-3′ (SEQ ID NO: 19)) and anti-HSVsiRNA (sigE (Bhuyan, P. K., et al., 2004); sense sequence5′-AATATACGAATCGTGTCTGTA-3′ (SEQ ID NO:20)). were synthesized by theoligo synthesis facility at the City of Hope (Duarte, Calif.).

T7 siRNAs

The T7 siRNAs were synthesized using the Silencer siRNA Construction kitfrom Ambion, Inc. according to the manufacturer's protocol. Totranscribe RNA in vitro, T7 primer I (5′-TAATACGACTCACTATA-3′ (SEQ IDNO: 15)) was hybridized with T7 primer II which contains the antisensesequence of each transcribed RNA and the tail sequence: 5′-CCCTATAGTGAGTCGTA-3′ (SEQ ID NO:16). The anti-EMCV siRNA is targeted to thesequence: 5′-GAT AGTGCCAGGGCGGGTACT-3′ (SEQ ID NO:21). The transcribedssRNA was used as T7 RNA without hybridization. For the CIP treatment,20 U of enzyme (NEB) was added to the siRNA after DNase and RNase T1digestion, and further incubated at 37° C. for 1 hour. siRNAs werecolumn purified using conditions recommended for the Silencer siRNAConstruction Kit.

EXAMPLE 7 Transfection

All transfection assays were done using Lipofectamine 2000 (Invitrogen).HEK293 or NIH3T3 cells at 50 to 60 percent confluency were transfectedwith each siRNAs using indicated concentrations. The siRNA andlipofectamine complex was simply added on top of existing growth media.

EXAMPLE 8 Viral Challenge Assay

For anti-HSV-1 or anti-polioviral assays, 60% confluent 293 cells onplates were transfected with siRNA or ssRNA using Lipofectamine 2000(Invitrogen). The following day (24 hours) the cells were infected withHSV-1 expressing the EGFP or Poliovirus Mahoney strain at a multiplicityof infection of 1 or 0.1, respectively. 24 hours post infection, theanti-HSV activity was measured by determining the EGFP level in theextract using a Fluorometer (Bio-Rad). To prepare the extract, the cellsin the 24 well plates were mixed with 200 μl of passive lysis buffer(Promega). For the anti-polioviral assay, the cells in each well werewashed with PBS three times to remove dead cells caused by the cytotoxiceffect of the virus and lysed by adding 200 μl of the lysis buffer.Total amount of protein was measured by the Bradford assay. Foranti-proliferation effect of T7 RNA or poly IC, 40% of the NIH3T3 cellsstably expressing EGFP gene was plated in 24 well plates on day 1. Thecells were transfected with T7 RNA or poly IC and harvested on day 5.Total cell numbers were determined by measuring EGFP levels using thefluorometer. For the anti-EMCV activity assay, cells were transfectedwith indicated amount of each RNAs on day 2. On day 3, the cells wereinfected with a 0.1 MOI of EMCV. Total anti-EMCV activity was measuredon day 5 or day 7. Total numbers of survived cells were determined bythe level of EGFP expression in the extracts after normalization to thevalue of each parallel sample from the anti-proliferation activityassay.

EXAMPLE 9 Viral RNA

Influenza virus A/PR/8/34 (PR8), subtype H1N1, a kind gift from Dr.Peter Palese, Mount Sinai School of Medicine, was grown for 48 hours in10-day-embryonated chicken eggs (Charles River laboratories, MA) at 37°C. 48 hours after virus inoculation, the allantoic fluid was harvestedand was centrifuged at 1300 rpm for 10 min. The supernatant was thenmixed with 25% sucrose in 0.1 M Tris (pH8.0) and ultracentrifuged at25,000 rpm for 2 hours using SW28. Following centrifugation, Trizol(Invitrogen) was added to the pellet and RNA purification was performedaccording to the manufacturer's instructions.

EXAMPLE 10 RT-PCR

Total RNA was purified using Stat60 and treated with 2 U of DNase(Promega) per ug of RNA for 20 min at 37° C. To detect Tlr3, Tlr7 andβ-actin mRNA using reverse transcriptase (RT) and PCR, first strand cDNAsynthesis was performed at 37° C. for 1 hour in a 30 μl reaction mixturecontaining 2 μg of total cellular RNA, 2 μmol of gene-specific primer(50 ng of random primers (invitrogen)), 0.5 mM each of DATP, dCTP, dTTPand dGTP, 3 mM MgCl₂, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 20 mM DTT, 5 URNasin RNase inhibitor (Promega) and 200 U M-MLV Reverse Transcriptase(Invitrogen). Reverse primers used for the PCR reaction (see below) wereused as gene-specific primers for first strand synthesis of Tlr3 andTlr7. Aliquots (5 μl) of the cDNA reaction mixture were used to amplifyTlr3, Tlr7 and β-actin sequences separately. The PCR reaction mixturesincluded 50 mM KCl, 10 mM Tris-HCl (pH 8.3 at 25° C.), 1.5 mM Mg(OAc)₂,0.2 mM each of dATP, dCTP, dTTP and dGTP, 15 pmol each of forward andreverse primers, and 2.5 U of Taq DNA polymerase (Eppendorf). Sequencesof forward and reverse Tlr3 primers were5′-AGATACAACGTAGCTGACTGCAGCCATTTG-3′ (SEQ ID NO:22) and5′-CTTCACTTCGCAACGCAAGGATTTTATTTT-3′ (SEQ ID NO:23). Sequences offorward and reverse Tlr7 primers were 5′-CATTCCCACTAACACCACCAATCTTACCCT-3′ (SEQ ID NO:24) and 5′-ATCCTGTGGTATCTCCAGAAGTTGGTTTCC-3′ (SEQID NO:25). Sequences of forward and reverse β-actin primers were5′-ACCAACTGGGACGA CATGGAGAAGATCTGG-3′ (SEQ ID NO:26) and5′-GCTGGGGTGTTGAAGGTCTCAAA CATGATC-3′ (SEQ ID NO:27). Thermal cyclingreactions were conducted at 95° C. for 30 seconds, 58° C. for 30 secondsand 72° C. for 1 minute. Aliquots were removed from the PCR reactionmixtures during the exponential phase of amplification after 25(β-actin) and 35 cycles (Tlr3 and Tlr7). Samples were resolved using 2%agarose gel electrophoresis. The same procedure was used for RT-PCR ofother genes using each pair of primers (for Stat 1, Ifi44, Tgtp,TNFaip3, Gadd4alpha).

EXAMPLE 11 Functional Inhibition Assay for TLR3

The procedure was followed as previously described (Matsumoto, M., etal., 2002). Anti-TLR2 and TLR3 antibodies were purchased fromeBioscience (San Diego, Calif.). Briefly, MRC-5 cells in 24 well pates(1×10⁵) were preincubated with 20 μg/ml of anti-TLR2 or anti-TLR3antibody for 1 hour at 37° C. The cells were transfected with either 5nM of T7 RNA or 500 ng of poly IC. The next day, interferon beta levelsin the media was determined by ELISA (RDI).

EXAMPLE 12 Microarray

Mouse oligonucleotides were purchased from Operon Technologies Inc.(Alameda, Calif.) and Sigma-Genosys (The Woodlands, Tex.), and wereinkjet-printed by Agilent Technologies (Palo Alto, Calif.). The 16Koligo array includes 13,536 Operon designed and synthesized probes (70mer), and 2,304 Compugen Ltd. (Jamesburg, N.J.) designed andSigma-Genosys synthesized probes (65 mer). The aminoallyl method wasused for the preparation of fluorescently labeled target samples.Briefly, both first and second strand cDNAs were synthesized byincubating 3 μg of total RNA with the T7 promoter primer (5′-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)₂₄-3′ (SEQ ID NO:28)) (Qiagen Inc.,Valencia, Calif.) followed by using SuperScript II (Invitrogen LifeTechnologies, Carlsbad, Calif.). Aminoallyl-UTP (aaUTP) labeledantisense RNA (aRNA) was synthesized by adding reagents to the 15 μl ofcDNA template in the following order: 4 μl of 75 mM ATP solution; 4 μlof 75 mM CTP solution; 4 μl of 75 mM GTP solution; 2 μl of 75 mM UTPsolution; 4 μl of 10× reaction buffer; 3 μl of 50 mM aaUTP (Ambion,Austin, Tex.); and 4 μl of MEGAscript T7 enzyme mix (Ambion). Thecoupling reaction was performed by mixing 10 μg of aRNA with 2 μl of 0.5M sodium bicarbonate, pH 9.5, along with 10 μl of mono-Cy3 or mono-Cy5solution (PerkinElmer, Inc.; Boston, Mass.), and adjusting the finalvolume to 20 μl/reaction. Three μg of each labeled aRNA target washybridized after being fragmented by mixing with fragmentation buffer(Agilent Technologies). After hybridization and washing, oligo arrayswere scanned by the Agilent Scanner G2505A (Agilent Technologies). Genesthat were saturated, non-uniform, or not significantly above background(below 2.6× standard deviation of background) in either channel wereremoved. The remaining values were used for the analysis.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Embodiments of this invention are described herein, including the bestmode known to the inventors for carrying out the invention. Variationsof those embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description. The inventors expectskilled artisans to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

BIBLIOGRAPHY

-   Ackermann, E. J., et al. Antisense modulation of novel    anti-apoptotic bcl-2-related proteins. U.S. Pat. No. 6,001,992    (1999).-   Agrawal, S., et al. Method of down-regulating gene expression. U.S.    Pat. No. 6,645,943 (2003).-   Akhtar, S. (ed.). Delivery Strategies for Antisense Oligonucleotide    Therapeutics, CRC Press, Boca Raton, Fla. (1995).-   Akhtar, S. and Juliano, R. L. Cellular uptake and intracellular fate    of antisense oligonucleotides. Trends Cell Biol, 2, 139 (1992).-   Alexopoulou, L., et al. Recognition of double-stranded RNA and    activation of NF-kappaB by Toll-like receptor 3. Nature 413, 732-8    (2001).-   Andino, R. RNAi puts a lid on virus replication. Nat. Biotechnol.    21, 629-630 (2003).-   Beigelman et al. Novel compositions for the delivery of negatively    charged molecules. PCT international published application No. WO    99/05094 (1999).-   Beutler, B. Inferences, questions and possibilities in Toll-like    receptor signalling. Nature 430, 257-63 (2004a).-   Beutler, B. Innate immunity: an overview. Mol Immunol 40, 845-59    (2004b).-   Boehme, K. W. and Compton, T. Innate sensing of viruses by toll-like    receptors. J Virol 78, 7867-73 (2004).-   Bridge, A. J., et a., Induction of an interferon response by RNAi    vectors in mammalian cells. Nature, 34(3), 263-264 (2003).-   Capodici, J., et al. Inhibition of HIV-1 infection by small    interfering RNA-mediated RNA interference. J. Immunol. 169,    5196-5201 (2002).-   Chou, M. Y., et al. hnRNP H is a component of a splicing enhancer    complex that activates a c-src alternative exon in neuronal cells.    Mol Cell Biol 19, 69-77 (1999).-   Der, S. D., et al. A double-stranded RNA-activated protein    kinase-dependent pathway mediating stress-induced apoptosis. Proc    Natl Acad Sci USA 94, 3279-83 (1997).-   Diebold, S. S., et al. Innate antiviral responses by means of    TLR7-mediated recognition of single-stranded RNA. Science 303,    1529-31 (2004).-   Dolken, G., et al. Immunofluorescent localization of glycogenolytic    and glycolytic enzyme proteins and of malate dehydrogenase isozymes    in cross-striated skeletal muscle and heart of the rabbit.    Histochemistry 43, 113-21 (1975).-   Donze, O. and Picard, D. RNA interference in mammalian cells using    siRNAs synthesized with T7 RNA polymerase. Nucleic Acids Res. 30,    e46 (2002).-   Draper et al. Method and reagent for inhibiting viral replication.    PCT international published application No. WO 93/23569 (1993).-   Elbashir, S. M., et al. Duplexes of 21-nucleotide RNAs mediate RNA    interference in cultured mammalian cells. Nature 411, 494-498    (2001).-   Elliott, G. and O'Hare, P. Live-cell analysis of a green fluorescent    protein-tagged herpes simplex virus infection. J. Virol. 73,    4110-4119 (1999).-   Fire, A., et al. Genetic inhibition by double-stranded RNA. U.S.    Pat. No. 6,506,559 (2003).-   Geiss, G. et al. A comprehensive view of regulation of gene    expression by double-stranded RNA-mediated cell signaling. J Biol    Chem 276, 30178-82 (2001).-   Gu, M. and Lima, C. D. Processing the message: structural insights    into capping and decapping mRNA. Curr Opin Struct Biol 15, 99-106    (2005).-   Hannon, G. J. RNA interference. Nature 418, 244-251 (2002).-   Heil, F. et al. Species-specific recognition of single-stranded RNA    via toll-like receptor 7 and 8. Science 303, 1526-9 (2004).-   Honda, A., et al. Identification of the 5′ terminal structure of    influenza virus genome RNA by a newly developed enzymatic method.    Virus Res. 55, 199-206 (1998).-   Hornung, V. et al. Sequence-specific potent induction of IFN-alpha    by short interfering RNA in plasmacytoid dendritic cells through    TLR7. Nat Med 11, 263-70 (2005).-   Kapadia, S. B., et al. Interference of hepatitis C virus RNA    replication by short interfering RNAs. Proc. Natl. Acad. Sci. USA    100, 2014-2018 (2003).-   Kariko, K., et al. mRNA is an endogenous ligand for Toll-like    receptor 3. J Biol Chem 279, 12542-50 (2004).-   Kim, D. H. and Rossi, J. J. Coupling of RNAi-mediated target    downregulation with gene replacement. Antisense Nucleic Acid Drug    Dev. 13, 151-155 (2003).-   Kim, D. H. et al. Interferon induction by siRNAs and ssRNAs    synthesized by phage polymerase. Nat Biotechnol 22, 321-5 (2004).-   Kim, D. H. et al. Synthetic dsRNA Dicer substrates enhance RNAi    potency and efficacy. Nat Biotechnol 23, 222-6 (2005).-   Klimuk, et al. Liposomal compositions for the delivery of nucleic    acid catalysts. PCT international published application No. WO    99/04819 (1999).-   Lamb, R. A. & Choppin, P. W. The gene structure and replication of    influenza virus. Annu Rev Biochem 52, 467-506 (1983).-   Lund, J. M. et al. Recognition of single-stranded RNA viruses by    Toll-like receptor 7. Proc Natl Acad Sci USA 101, 5598-603 (2004).-   Malmgaard, L. Induction and regulation of IFNs during viral    infections. J Interferon Cytokine Res 24, 439-54 (2004).-   Matsumoto, M., et al. Establishment of a monoclonal antibody against    human Toll-like receptor 3 that blocks double-stranded RNA-mediated    signaling. Biochem Biophys Res Commun 293, 1364-9 (2002).-   Marcus, P. I., Interferon, 3 (ed. Gresser, I.) 115-180 (Academic    Press, London, 1983).-   Montgomery, M. K. RNA Interference, Editing, and Modification:    Methods and Protocols. Methods in Molecular Biology, 265, 3-21,    (2004).-   Samuel, C. E. Antiviral actions of interferons. Clin. Microbiol.    Rev. 14, 778-809 (2001).-   Samuel, C. E. Knockdown by RNAi-proceed with caution. Nature    Biotechnology, 22(3), 280-82 (2004).-   Sledz, C. A., et al. Activation of the interferon system by    short-interfering RNAs. Nature Cell Biology, 5(9), 834-39 (2003).-   Sohail, M., et al. A simple and cost-effective method for producing    small interfering RNAs with high efficacy. Nucleic Acids Res. 31,    e38 (2003).-   Stewart, W. E., II., The Interferon System (Springer, N.Y., 1979).-   Stojdl, D. F. et al. Exploiting tumor-specific defects in the    interferon pathway with a previously unknown oncolytic virus. Nat.    Med. 6, 821-825 (2000).-   Sullivan et al. Method and reagent for treatment of animal diseases.    PCT international published application No. WO 94/02595 (1994).-   Thompson, J. et al. Nucleic acid molecules with novel chemical    compositions capable of modulating gene expression. U.S. Pat. No.    6,673,611 (2004).-   Wang, L. et al. Mapping oligonucleotides of Rous sarcoma virus RNA    that segregate with polymerase and group-specific antigen markers in    recombinants. Proc. Natl. Acad. Sci. USA 73, 3952-3956 (1976).-   Wei, C. and Moss, B. 5′-Terminal capping of RNA by    guanylyltransferase from HeLa cell nuclei. Proc Natl Acad Sci USA    74, 3758-61 (1977).

What is claimed is:
 1. A method of inducing an anti-viral response in acell, comprising introducing into a cell an effective amount of a siRNAmolecule, wherein the siRNA molecule is double stranded, wherein thesiRNA molecule has a 5′-triphosphate on each strand, wherein the siRNAmolecule is targeted to a viral gene, wherein the siRNA molecule inducesinterferon which mediates an anti-viral response and wherein the siRNAmolecule reduces expression of the targeted viral gene.
 2. The method ofclaim 1, wherein each strand of the siRNA molecule is produced in vitroby a phage polymerase.
 3. The method of claim 2, wherein the phagepolymerase is T7 RNA polymerase.